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The anti-centrosome monoclonal antibody 6C6 reacts with a plasma membrane-associated polypeptide of 77 kDa fromNicotiana tabacum pollen tubes

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Summary

In the pollen and pollen tube of higher plants, the distribution of the microtubular cytoskeleton has been extensively studied. Even though the pattern of microtubules is known, one of the most remarkable deficiencies is the absence of data on the localization of microtubule-nucleation sites in the pollen tubes. In order to get insights about the localization of centrosome-like structures in the pollen tube ofNicotiana tabacum L., we have used the monoclonal antibody 6C6 to search for pericentriolar antigen(s). The antibody was initially raised against a component of animal centrosomes and has been already employed to locate centrosomal structures in other plant cell types. By immunoblotting analysis, a polypeptide of Mr 77,000 was identified specifically in the membrane-associated protein fraction of the pollen tube, and is absent from the soluble protein pool. Immunofluorescence observations have shown the polypeptide to be located in the apical part of the pollen tube (about 40–50 μm from the tip) in association with the cortical area. A purified plasma membrane fraction from the growing pollen tubes has been obtained, using H+-ATPase activity as an organelle marker. The plasma membrane fraction was shown to be enriched in the Mr 77,000 polypeptide, which can be extracted from membranes by treatment with the detergent CHAPS at a concentration of 0.5%. These data open new research perspectives on the localization and analysis of putative cortical microtubule nucleation sites in the pollen tube.

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Abbreviations

ATP:

adenosine-5′-triphosphate

CHAPS:

3-[(3-cholamidopropyl)-dimethylammonio]-propanesulfonate

DTT:

dithiothreitol

EDTA:

ethylenediaminetetracetic acid

EGTA:

ethylene glycolbis(β-amino-ethyl ether) N,N,N′,N′-tetraacetic acid

HEPES:

4-(2-hydroxyethyl)-1-piperazineethane sulphonic acid

MES:

2-(N-morpholino)ethane sulphonic acid

MT:

microtubule

SDS-PAGE:

sodium-dodecyl-sulphate polyacrylamide gel electrophoresis

PMSF:

phenylmethyl-sulphonyl-fluoride

TAME:

tosyl-arginine-methylester

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Authors and Affiliations

  1. Dipartimento Biologia Ambientale, Università di Siena, via P.A. Mattioli 4, 1-53100, Siena, Italy

    G. Cai, A. Moscatelli, C. Del Casino, M. Mazzi & M. Cresti

  2. Départment de Biologie Moléculaire et Structurale, Laboratoire du Cytosquelette, INSERM U366, Centre d'Etudes Nucléaires, Grenoble

    V. Chevrier

  3. Dipartimento Scienze Ambientali, Università delia Tuscia, Viterbo

    A. Tiezzi

Authors
  1. G. Cai
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  2. A. Moscatelli
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  3. C. Del Casino
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  4. V. Chevrier
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  5. M. Mazzi
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  6. A. Tiezzi
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  7. M. Cresti
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Cai, G., Moscatelli, A., Del Casino, C. et al. The anti-centrosome monoclonal antibody 6C6 reacts with a plasma membrane-associated polypeptide of 77 kDa fromNicotiana tabacum pollen tubes. Protoplasma 190, 68–78 (1996). https://doi.org/10.1007/BF01281195

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  • DOI: https://doi.org/10.1007/BF01281195

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